CN115452973B - Method for detecting ethyl chloroformate in thiamphenicol hydrochloride glycine ester - Google Patents
Method for detecting ethyl chloroformate in thiamphenicol hydrochloride glycine ester Download PDFInfo
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- RIFGWPKJUGCATF-UHFFFAOYSA-N ethyl chloroformate Chemical compound CCOC(Cl)=O RIFGWPKJUGCATF-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 21
- -1 thiamphenicol hydrochloride glycine ester Chemical class 0.000 title claims description 9
- 229960003053 thiamphenicol Drugs 0.000 title description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 title description 2
- 239000004471 Glycine Substances 0.000 title description 2
- DKXJDBJNAXSJDH-MHDYBILJSA-N [(2r,3r)-2-[(2,2-dichloroacetyl)amino]-3-hydroxy-3-(4-methylsulfonylphenyl)propyl] 2-aminoacetate;hydron;chloride Chemical compound Cl.CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](COC(=O)CN)NC(=O)C(Cl)Cl)C=C1 DKXJDBJNAXSJDH-MHDYBILJSA-N 0.000 claims abstract description 36
- 238000001212 derivatisation Methods 0.000 claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 16
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims abstract description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 96
- 239000000243 solution Substances 0.000 claims description 77
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 63
- 238000002156 mixing Methods 0.000 claims description 26
- 239000011550 stock solution Substances 0.000 claims description 26
- 239000000523 sample Substances 0.000 claims description 21
- LQERIDTXQFOHKA-UHFFFAOYSA-N nonadecane Chemical compound CCCCCCCCCCCCCCCCCCC LQERIDTXQFOHKA-UHFFFAOYSA-N 0.000 claims description 20
- 239000013558 reference substance Substances 0.000 claims description 20
- 230000035945 sensitivity Effects 0.000 claims description 20
- 239000003795 chemical substances by application Substances 0.000 claims description 19
- 238000012360 testing method Methods 0.000 claims description 17
- 239000012490 blank solution Substances 0.000 claims description 16
- 239000012488 sample solution Substances 0.000 claims description 16
- 239000012159 carrier gas Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 239000012086 standard solution Substances 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- RTDCJKARQCRONF-UHFFFAOYSA-N N-Nitrosomethylethylamine Chemical compound CCN(C)N=O RTDCJKARQCRONF-UHFFFAOYSA-N 0.000 claims description 3
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000004949 mass spectrometry Methods 0.000 claims description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 125000001664 diethylamino group Chemical group [H]C([H])([H])C([H])([H])N(*)C([H])([H])C([H])([H])[H] 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 229960002355 thiamphenicol glycinate Drugs 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 6
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 4
- 238000003908 quality control method Methods 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- CYTYCFOTNPOANT-UHFFFAOYSA-N Perchloroethylene Chemical compound ClC(Cl)=C(Cl)Cl CYTYCFOTNPOANT-UHFFFAOYSA-N 0.000 description 15
- 238000010790 dilution Methods 0.000 description 12
- 239000012895 dilution Substances 0.000 description 12
- 239000012085 test solution Substances 0.000 description 7
- 238000011835 investigation Methods 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 239000012088 reference solution Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- OTVAEFIXJLOWRX-NXEZZACHSA-N thiamphenicol Chemical class CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CO)NC(=O)C(Cl)Cl)C=C1 OTVAEFIXJLOWRX-NXEZZACHSA-N 0.000 description 4
- 238000005259 measurement Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000012946 outsourcing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- JIGUQPWFLRLWPJ-UHFFFAOYSA-N Ethyl acrylate Chemical class CCOC(=O)C=C JIGUQPWFLRLWPJ-UHFFFAOYSA-N 0.000 description 1
- VAJCYQHLYBTSHG-UHFFFAOYSA-N ethyl n,n-diethylcarbamate Chemical compound CCOC(=O)N(CC)CC VAJCYQHLYBTSHG-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/025—Gas chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention belongs to the technical field of medicine analysis, and particularly relates to a method for detecting ethyl chloroformate in thiamphenicol glycinate, which is convenient, efficient and accurate for solving the problem of detecting ethyl chloroformate in thiamphenicol glycinate, and the method is characterized in that a derivatization reagent diethylamine is used for carrying out pre-column derivatization, N-diethyl ethoxyformamide is generated after derivatization, and the detection is carried out in GC-MS/MS, so that the content of ethyl chloroformate in thiamphenicol glycinate can be detected, the medication safety is effectively ensured, the quality control of thiamphenicol glycinate is convenient, and the method is convenient, efficient and accurate, completely accords with the guidelines verified by the Chinese pharmacopoeia method in the aspects of system applicability, repeatability, specificity and accuracy and can be used for the quality control of thiamphenicol glycinate hydrochloride.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a detection method of ethyl chloroformate in thiamphenicol glycinate hydrochloride.
Background
Thiamphenicol glycinate hydrochloride is a derivative of thiamphenicol, is a similar substance of chloramphenicol, has an antibacterial spectrum and an antibacterial effect similar to those of chloramphenicol, has good water solubility, and can rapidly release thiamphenicol to play a role after the pH value of the solution is increased or the solution is subjected to the action of in vivo enzymes, so that the clinical application of thiamphenicol is widened, and the disadvantage of poor water solubility of thiamphenicol is overcome.
Ethyl chloroformate, chemical structure:the thiamphenicol glycinate hydrochloride is a synthetic material of thiamphenicol glycinate hydrochloride, and ethyl chloroformate acyl halide genotoxic impurities possibly remain in the thiamphenicol glycinate hydrochloride, so that the content of the thiamphenicol glycinate hydrochloride needs to be strictly controlled in the production process of the thiamphenicol glycinate hydrochloride, the maximum daily dosage of the thiamphenicol glycinate hydrochloride in the thiamphenicol glycinate hydrochloride tablet is 1g/day by referring to the ICH M7 toxicology threshold TTC=1.5 mu g/day, and the limit of the ethyl chloroformate is calculated by a formula of limit=TTC/maximum daily dosage.
The invention discloses a detection method for detecting ethyl chloroformate in thiamphenicol glycinate hydrochloride by adopting GC-MS/MS, which is reported in the literature at present, adopts a derivatization reagent diethylamine to carry out pre-column derivatization, generates N, N-diethyl ethoxyformamide after derivatization, uses the GC-MS/MS method to detect, and provides a convenient, efficient and accurate detection method for solving the problem of detecting ethyl chloroformate in thiamphenicol glycinate hydrochloride, and the method can detect the content of ethyl chloroformate in thiamphenicol glycinate hydrochloride, thereby effectively guaranteeing medication safety and facilitating quality control of thiamphenicol glycinate hydrochloride.
Disclosure of Invention
The invention provides a detection method for detecting ethyl chloroformate in thiamphenicol glycinate hydrochloride by adopting GC-MS/MS, which is convenient, efficient and accurate, and can be used for detecting the content of ethyl chloroformate in thiamphenicol glycinate hydrochloride, thereby effectively guaranteeing the medication safety and facilitating the quality control of thiamphenicol glycinate hydrochloride.
In order to achieve the above purpose, the present invention provides the following technical solutions:
an inspection method of ethyl chloroformate in thiamphenicol glycinate hydrochloride, the inspection method comprising the following steps:
(1) Preparing a solution, namely preparing a blank solution, a sensitivity solution, a reference substance solution and a test sample solution respectively;
(2) The measuring method comprises the following steps: determining the content of ethyl chloroformate in thiamphenicol glycinate hydrochloride by adopting GC-MS/MS, respectively adding a blank solution, a sensitivity solution, a reference substance solution and a test sample solution after the system is stable, and recording a chromatogram;
the chromatographic conditions are as follows: capillary column with 6% cyanopropyl/phenyl and 94% polydimethylsiloxane as fixing solution, column temperature: the initial column temperature is 80 ℃, kept for 2min, heated to 140 ℃ at the speed of 30 ℃/min, kept for 1min, heated to 220 ℃ at the speed of 50 ℃/min, and kept for 7min; sample inlet temperature: 250 ℃, carrier gas flow rate: 2.0ml/min, split ratio: 1:1, carrier gas: he, sample injection amount: 1 μl of needle wash A: dichloromethane, needle washing liquid B: acetonitrile;
the mass spectrometry conditions were as follows: ion source temperature: 230 ℃, gas chromatography-mass spectrometry interface temperature: ion source voltage at 240 ℃): 70eV, detection mode: MRM, gain factor: 5, solvent excision time: 0-3.5 min, mass rod temperature: 150 ℃, MRM parameter set:
further, the blank solution: taking a centrifuge tube, adding a derivatizing agent, acetonitrile and an ISTD solution, uniformly mixing, adding dichloromethane, uniformly mixing, standing for 10min, and taking a lower layer;
the ISTD solution is: transferring a proper amount of N-nitrosomethylethylamine standard solution into a measuring flask, adding acetonitrile to dilute to a scale, and shaking uniformly;
the control solution: placing a proper amount of ethyl chloroformate reference substance into a measuring flask, adding acetonitrile, diluting to a scale, and shaking uniformly to prepare reference substance stock solution with proper concentration; taking a centrifuge tube, adding the derivatization agent, the reference substance stock solution and the ISTD solution, uniformly mixing, adding dichloromethane, uniformly mixing, standing for 10min, and taking the lower layer;
the sensitivity solution: taking a proper amount of reference substance stock solution, placing the reference substance stock solution into a measuring flask, adding acetonitrile to dilute to a scale, and shaking uniformly to prepare a sensitivity stock solution with proper concentration; taking a centrifuge tube, adding a derivatization agent, a sensitivity stock solution and an ISTD solution, uniformly mixing, adding dichloromethane, uniformly mixing, standing for 10min, and taking a lower layer;
the test solution: taking a proper amount of a test sample, precisely weighing, placing into a centrifuge tube, adding a derivatization agent, acetonitrile and an ISTD solution, shaking until the test sample is completely dissolved, adding dichloromethane, uniformly mixing, standing for 10min, and taking out the lower layer;
the chromatographic column is Agilent DB-624, 30mX0.32mm, 1.8 mu m or capillary column with equivalent efficiency.
Further, the measurement method of the present invention comprises the steps of:
(1) Preparing a solution:
dilution liquid: acetonitrile;
derivatization agent: transferring 10.0ml diethylamine into a 100ml measuring flask, adding water to dilute to scale, and shaking uniformly;
ISTD solution: transferring NEMA standard solution (1000 mug/ml) into a 250 mug-10 ml measuring flask, and adding acetonitrile to dilute to a scale; and precisely measuring 500 μl of the solution, placing in a 100ml measuring flask, adding acetonitrile for dilution to the scale, and shaking uniformly. (concentration: NEMA 125 ng/ml)
Ethyl chloroformate stock solution: taking about 50mg of ethyl chloroformate reference substance, precisely weighing, placing into a 25ml measuring flask, diluting with acetonitrile to scale, and shaking; and precisely measuring 150 μl of the solution, placing in a 20ml measuring flask, adding acetonitrile for dilution to the scale, and shaking uniformly. (concentration: ethyl chloroformate 15. Mu.g/ml)
Control stock solution: precisely measuring 500 μl of ethyl chloroformate stock solution, placing in a 100ml measuring flask, adding acetonitrile for dilution to scale, and shaking uniformly. (concentration: ethyl chloroformate 75 ng/ml)
Sensitivity stock solution: precisely measuring 3.0ml of control stock solution, placing into a 10ml measuring flask, adding acetonitrile for dilution to scale, and shaking uniformly. (concentration: 22.5 ng/ml)
Blank solution (internal standard added): taking a 15ml centrifuge tube, adding 1.0ml of a derivatizing agent, 1.0ml of acetonitrile and 1.0ml of an ISTD solution, uniformly mixing, adding 8.0ml of dichloromethane, uniformly mixing, standing for 10min, and taking a lower layer. (concentration: NEMA 12.5 ng/ml)
Control solution: taking a 15ml centrifuge tube, adding 1.0ml of a derivatization agent, 1.0ml of a reference substance stock solution and 1.0ml of an ISTD solution, uniformly mixing, adding 8.0ml of dichloromethane, uniformly mixing, standing for 10min, and taking a lower layer. (concentration: ethyl chloroformate 7.5 ng/ml; NEMA 12.5 ng/ml)
Sensitivity solution: taking a 15ml centrifuge tube, adding 1.0ml of a derivatization agent, 1.0ml of a sensitivity stock solution and 1.0ml of an ISTD solution, uniformly mixing, adding 8.0ml of dichloromethane, uniformly mixing, standing for 10min, and taking a lower layer. ( Concentration: ethyl chloroformate 2.25 ng/ml; NEMA 12.5ng/ml )
Test solution: about 50mg of a test sample is taken, precisely weighed, placed in a 15ml centrifuge tube, 1.0ml of a derivatization agent, 1.0ml of acetonitrile and 1.0ml of an ISTD solution are added, the test sample is shaken until the test sample is completely dissolved, 8.0ml of dichloromethane is added, the mixture is uniformly mixed, and the mixture is kept stand for 10min, and the lower layer is taken. ( Concentration: thiamphenicol glycinate hydrochloride 5mg/ml; NEMA 12.5ng/ml )
(2) The measuring method comprises the following steps:
after the system is stable, 1-3 needles of blank solution (with internal standard added), 1 needle of sensitivity solution, 6 needles of reference solution, 1 needle of blank solution (with internal standard added), 1 needle of test solution and record spectrograms.
Results (ppm) =
Wherein: r is R U : a measured value of a ratio (RR) of the peak area of the ethyl chloroformate derivative to the peak area of the internal standard in the sample solution map; r is R S :6 is an average value of the ratio (RR) of the peak area of the ethyl chloroformate derivative to the peak area of the internal standard substance in the reference substance solution map; c (C) S : concentration of ethyl chloroformate in control solution (ng/ml); n: dilution factor of test sample, 10; m: sample amount, mg.
Limit:
the method for detecting ethyl chloroformate in thiamphenicol glycinate hydrochloride further comprises the step of method verification before detection, wherein the method verification is carried out according to chromatographic conditions of formal detection, and the measurement result is as follows:
the invention discloses a method for detecting ethyl chloroformate in thiamphenicol glycinate, which aims to solve the problem of detecting ethyl chloroformate in thiamphenicol glycinate, and can detect the content of ethyl chloroformate in thiamphenicol glycinate conveniently, efficiently and accurately, so that the medication safety is effectively ensured.
Drawings
FIG. 1 is a blank solution pattern of ethyl chloroformate in thiamphenicol glycinate hydrochloride;
FIG. 2 is a graph of ethyl chloroformate blank (with internal standard) solution in thiamphenicol glycinate hydrochloride;
FIG. 3 is a graph of a sample solution of ethyl chloroformate in thiamphenicol glycinate hydrochloride;
FIG. 4 is a graph of ethyl chloroformate control solution in thiamphenicol glycinate hydrochloride;
FIG. 5 is a graph of a selective solution of ethyl chloroformate in thiamphenicol glycinate hydrochloride.
Description of the embodiments
The invention is further illustrated by the following examples, which are not intended to be limiting.
Example 1
(1) Preparing a solution:
acetonitrile: HPLC, water: ultrapure water, diethylamine: AR and above, dichloromethane: HPLC, ethyl chloroformate control: outsourcing, N-Nitrosomethylethylamine (NEMA) reference (1000 mug/ml), internal standard reference: outsourcing
Dilution liquid: acetonitrile;
derivatization agent: transferring 10.0ml diethylamine into a 100ml measuring flask, adding water to dilute to scale, and shaking uniformly;
ISTD solution: transferring NEMA standard solution (1000 mug/ml) into a 250 mug-10 ml measuring flask, and adding acetonitrile to dilute to a scale; and precisely measuring 500 μl of the solution, placing in a 100ml measuring flask, adding acetonitrile for dilution to the scale, and shaking uniformly. (concentration: NEMA 125 ng/ml)
Ethyl chloroformate stock solution: taking about 50mg of ethyl chloroformate reference substance, precisely weighing, placing into a 25ml measuring flask, diluting with acetonitrile to scale, and shaking; and precisely measuring 150 μl of the solution, placing in a 20ml measuring flask, adding acetonitrile for dilution to the scale, and shaking uniformly. (concentration: ethyl chloroformate 15. Mu.g/ml)
Control stock solution: precisely measuring 500 μl of ethyl chloroformate stock solution, placing in a 100ml measuring flask, adding acetonitrile for dilution to scale, and shaking uniformly. (concentration: ethyl chloroformate 75 ng/ml)
Sensitivity stock solution: precisely measuring 3.0ml of control stock solution, placing into a 10ml measuring flask, adding acetonitrile for dilution to scale, and shaking uniformly. (concentration: 22.5 ng/ml)
Blank solution (internal standard added): taking a 15ml centrifuge tube, adding 1.0ml of a derivatizing agent, 1.0ml of acetonitrile and 1.0ml of an ISTD solution, uniformly mixing, adding 8.0ml of dichloromethane, uniformly mixing, standing for 10min, and taking a lower layer. (concentration: NEMA 12.5 ng/ml)
Control solution: taking a 15ml centrifuge tube, adding 1.0ml of a derivatization agent, 1.0ml of a reference substance stock solution and 1.0ml of an ISTD solution, uniformly mixing, adding 8.0ml of dichloromethane, uniformly mixing, standing for 10min, and taking a lower layer. (concentration: ethyl chloroformate 7.5 ng/ml; NEMA 12.5 ng/ml)
Sensitivity solution: taking a 15ml centrifuge tube, adding 1.0ml of a derivatization agent, 1.0ml of a sensitivity stock solution and 1.0ml of an ISTD solution, uniformly mixing, adding 8.0ml of dichloromethane, uniformly mixing, standing for 10min, and taking a lower layer. ( Concentration: ethyl chloroformate 2.25 ng/ml; NEMA 12.5ng/ml )
Test solution: about 50mg of a test sample is taken, precisely weighed, placed in a 15ml centrifuge tube, 1.0ml of a derivatization agent, 1.0ml of acetonitrile and 1.0ml of an ISTD solution are added, the test sample is shaken until the test sample is completely dissolved, 8.0ml of dichloromethane is added, the mixture is uniformly mixed, and the mixture is kept stand for 10min, and the lower layer is taken. (concentration: thiamphenicol glycinate hydrochloride 5mg/ml; NEMA 12.5 ng/ml).
The chromatographic conditions were as follows: gas chromatograph, autosampler, triple quadrupole mass spectrometer equipped with EI ion source and electronic analytical balance
Chromatographic column: capillary columns with 6% cyanopropyl/phenyl and 94% polydimethylsiloxane as fixatives (e.g., agilent DB-624, 30mX0.32mm, 1.8 μm or equivalent capillary columns)
Column temperature: the initial column temperature was 80℃for 2min, heated to 140℃at a rate of 30℃per minute for 1min, and then heated to 220℃at a rate of 50℃per minute for 7min.
Sample inlet temperature: carrier gas flow rate at 250 c: 2.0ml/min
Split ratio: 1:1 carrier gas: he (He)
Sample injection amount: 1 μl of needle wash A: dichloromethane (dichloromethane)
Needle washing liquid B: acetonitrile
Mass spectrometry conditions
Ion source temperature: gas chromatography-mass spectrometry interface temperature at 230 ℃): 240 DEG C
Ion source voltage: 70eV detection mode: MRM (MRM)
Gain factor: 5. solvent excision time: 0 to 3.5min
Mass bar temperature: 150 DEG C
MRM parameter settings:
(2) The measuring method comprises the following steps:
after the system is stable, 1-3 needles of blank solution (with internal standard) are added, 1 needle of sensitivity solution, 6 needles of reference solution, 1 needle of blank solution (with internal standard) and 1 needle of test solution are added, and the spectrogram is recorded.
Results (ppm) =
Wherein: r is R U : a measured value of a ratio (RR) of the peak area of the ethyl chloroformate derivative to the peak area of the internal standard in the sample solution map; r is R S :6 is an average value of the ratio (RR) of the peak area of the ethyl chloroformate derivative to the peak area of the internal standard substance in the reference substance solution map; c (C) S : concentration of ethyl chloroformate in control solution (ng/ml); n: dilution factor of test sample, 10; m: sample amount, mg.
Limit:
example 2: system applicability
The system applicability was achieved by evaluating the signal to noise ratio (S/N) of the ethyl chloroformate derivative in the sensitivity solution and RSD of the ratio (RR) of the peak area of the ethyl chloroformate derivative to the peak area of the internal standard in the 6-needle control solution. The S/N value of the ethyl chloroformate derivative in the sensitivity solution is required to be not less than 10; the RSD of the ratio (RR) of the peak area of the ethyl chloroformate derivative to the peak area of the internal standard in the 6-needle control solution should be not more than 10.0%. To confirm the system applicability during the sequence run, every 8 hours or at the end of the sequence during the verification process, a control solution is added, and the RSD of the ratio (RR) of the peak area of the ethyl chloroformate derivative to the peak area of the internal standard in the 6-needle control solution is required to be no more than 10.0%; if the range is out, an evaluation investigation should be made.
Example 3: specialization of
The specificity is realized by measuring whether a blank solution has interference on ethyl chloroformate derivative and an internal standard (NEMA), wherein the blank solution (added with the internal standard) has interference on ethyl chloroformate derivative detection, the separation degree between a peak and an adjacent peak of the ethyl chloroformate derivative in the selective solution is required to be not interfered on both the ethyl chloroformate derivative and the internal standard (NEMA), the blank solution (added with the internal standard) has no interference on the ethyl chloroformate derivative detection, and the separation degree between the peak and the adjacent peak of the ethyl chloroformate derivative in the selective solution is required to be not less than 1.5.
Example 4: precision of
Repeatability: repeatability is achieved by measuring the RSD of ethyl chloroformate measurements in 6 parts of the test solution (labeled), requiring that the RSD of ethyl chloroformate measurements in 6 parts of the test solution (labeled) should be no greater than 20.0%.
Example 5: limit of detection and limit of quantification
The detection limit is obtained by detecting that the ratio of the response signal to the noise is not less than 3:1; the quantitative limit is determined by detecting that the ratio of the response signal to the noise is not less than 10: 1. At the quantitative concentration limiting level, repeatedly examining 6 parts of quantitative limiting solution, wherein in 6 parts of LOQ solution, the RSD of the ratio (RR) of the peak area of the ethyl chloroformate derivative with unit concentration to the peak area of the internal standard substance is required to be not more than 20.0%; the LOQ ethyl chloroformate should not be more than 0.5ppm, and the S/N of the ethyl chloroformate derivative in the LOQ solution is more than or equal to 10; the S/N of the ethyl chloroformate derivative in the LOD solution is more than or equal to 3, and the LOD is less than LOQ.
Example 6: linearity and range
Uniformly taking 6 points within the limit concentration range of LOQ concentration to 150 percent, taking the concentration as the abscissa and taking methyl chlorideThe ratio (RR) of the peak area of the ethyl acrylate derivative to the peak area of the internal standard is plotted as the ordinate. It is required that ethyl chloroformate should be linear in the range of LOQ concentration to 150% limit concentration, and the square R of the linear correlation coefficient 2 The absolute value of the y-axis intercept should be not less than 0.99 and within 25% of the ratio (RR) of the peak area of the 100% strength ethyl chloroformate derivative to the peak area of the internal standard.
Example 7: accuracy of
Accuracy means that the measured result is close to the true value or the reference value by measuring the recovery rate of standard solutions with different concentrations in the solution of the test sample, the recovery rate of ethyl chloroformate in the solution with accuracy required to add LOQ concentration, 100% limit concentration and 150% limit concentration is 70.0% -130.0%, and the total RSD (n=9) of the recovery rate is not more than 20.0%.
Example 8: durability of
And (3) examining the rule of time variation of detection results after the control solution, the sample solution and the selective solution are placed for a period of time at room temperature, and providing a basis for the placement time of the control solution and the sample solution during detection.
The requirements are: if the ethyl chloroformate is detected in the sample solution for 0hr, the sample solution is placed at room temperature for a period of time, the measured result change value is within 20% of the limit of the ethyl chloroformate, and no obvious change trend exists, and the sample solution is stable during room temperature investigation; if the ethyl chloroformate is not detected in the sample solution for 0hr, the sample solution is still not detected in the ethyl chloroformate after being placed at room temperature for a period of time, and the sample solution is stable during room temperature investigation;
compared with the reference solution for 0hr, the reference solution is placed at room temperature for a period of time, the recovery rate of ethyl chloroformate is 80.0% -120.0%, no obvious change trend exists, and the reference solution is stable during investigation;
the selective solution is placed at room temperature for a period of time, the recovery rate of ethyl chloroformate is 80.0% -120.0%, no obvious change trend exists, and the selective solution is stable during investigation.
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Claims (2)
1. The method for detecting ethyl chloroformate in thiamphenicol glycinate hydrochloride is characterized by comprising the following steps of:
(1) Preparing a solution, namely preparing a blank solution, a sensitivity solution, a reference substance solution and a test sample solution respectively;
(2) The measuring method comprises the following steps: determining the content of ethyl chloroformate in thiamphenicol glycinate hydrochloride by adopting GC-MS/MS, respectively adding a blank solution, a sensitivity solution, a reference substance solution and a test sample solution after the system is stable, and recording a chromatogram;
the chromatographic conditions were as follows:
chromatographic column: capillary column with 6% cyanopropyl/phenyl and 94% polydimethylsiloxane as fixing solution, column temperature: the initial column temperature is 80 ℃, kept for 2min, heated to 140 ℃ at the speed of 30 ℃/min, kept for 1min, heated to 220 ℃ at the speed of 50 ℃/min, and kept for 7min; sample inlet temperature: 250 ℃, carrier gas flow rate: 2.0ml/min, split ratio: 1:1, carrier gas: he, sample injection amount: 1 μl of needle wash A: dichloromethane, needle washing liquid B: acetonitrile;
the mass spectrometry conditions were as follows: ion source temperature: 230 ℃, gas chromatography-mass spectrometry interface temperature: ion source voltage at 240 ℃): 70eV, detection mode: MRM, gain factor: 5, solvent excision time: 0-3.5 min, mass rod temperature: 150 ℃, MRM parameter set:
the preparation method of the sample solution comprises the following steps: taking a proper amount of a test sample, precisely weighing, placing into a centrifuge tube, adding a derivatization agent, acetonitrile and an ISTD solution, shaking until the test sample is completely dissolved, adding dichloromethane, uniformly mixing, standing for 10min, and taking out the lower layer;
the derivatizing agent is diethylamine.
2. The method of claim 1, wherein the blank solution: taking a centrifuge tube, adding a derivatizing agent, acetonitrile and an ISTD solution, uniformly mixing, adding dichloromethane, uniformly mixing, standing for 10min, and taking a lower layer;
the ISTD solution is: transferring a proper amount of N-nitrosomethylethylamine standard solution into a measuring flask, adding acetonitrile to dilute to a scale, and shaking uniformly;
the control solution: placing a proper amount of ethyl chloroformate reference substance into a measuring flask, adding acetonitrile, diluting to a scale, and shaking uniformly to prepare reference substance stock solution with proper concentration; taking a centrifuge tube, adding the derivatization agent, the reference substance stock solution and the ISTD solution, uniformly mixing, adding dichloromethane, uniformly mixing, standing for 10min, and taking the lower layer;
the sensitivity solution: taking a proper amount of reference substance stock solution, placing the reference substance stock solution into a measuring flask, adding acetonitrile to dilute to a scale, and shaking uniformly to prepare a sensitivity stock solution with proper concentration; taking a centrifuge tube, adding a derivatization agent, a sensitivity stock solution and an ISTD solution, uniformly mixing, adding dichloromethane, uniformly mixing, standing for 10min, and taking a lower layer;
the chromatographic column is Agilent DB-624, 30mX0.32mm, 1.8mu m.
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| CN102012406A (en) * | 2010-09-26 | 2011-04-13 | 北京四环科宝制药有限公司 | Quality detection method of thiamphenicol glycinate hydrochloride |
| CN112114054A (en) * | 2019-06-21 | 2020-12-22 | 万特制药(海南)有限公司 | Method for determining genetic toxic substance ethyl chloroformate in loratadine by gas chromatography |
| WO2021022876A1 (en) * | 2019-08-06 | 2021-02-11 | 特丰制药有限公司 | Method for determining halogenated acid content in chloral hydrate or preparation thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102012406A (en) * | 2010-09-26 | 2011-04-13 | 北京四环科宝制药有限公司 | Quality detection method of thiamphenicol glycinate hydrochloride |
| CN112114054A (en) * | 2019-06-21 | 2020-12-22 | 万特制药(海南)有限公司 | Method for determining genetic toxic substance ethyl chloroformate in loratadine by gas chromatography |
| WO2021022876A1 (en) * | 2019-08-06 | 2021-02-11 | 特丰制药有限公司 | Method for determining halogenated acid content in chloral hydrate or preparation thereof |
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| 气相色谱法测定氯乙酸乙酯的含量;谭宁会;何姣;;广州化工;20180408(07);全文 * |
| 气相色谱法测定甘氨酰胺盐酸盐中氯乙酸甲酯的含量;李才岛;韦华;;海南医学;20151125(22);全文 * |
| 连秋燕 ; .1,2-二氯乙烷及其降解产物的GC-MS法测定.印染.2015,(01),全文. * |
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